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HSP27 immunohistochemical expression in colorectal carcinoma

does it have a prognostic role?

O.K., Rizk; M.A., El-Rashidy

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Egyptian Journal of Pathology: July 2016 - Volume 36 - Issue 1 - p 71-75
doi: 10.1097/01.XEJ.0000484377.13917.3d
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Abstract

Introduction

Colorectal cancer (CRC) is the most common type of gastrointestinal cancer and the second most frequently diagnosed malignancy in adults. Computed tomography and MRI are the modalities used most frequently for staging. Surgical resection may be curative, although the 5-year survival rate is 40–50% (Astin et al., 2011). It is a multifactorial disease process, with etiology encompassing genetic factors, environmental exposures (including diet), and inflammatory conditions of the digestive tract (Thota et al., 2014). There is a clear consensus of opinion that tumors that have spread to regional lymph nodes should receive adjuvant chemotherapy. Tumors that have breached the muscularis mucosae but have not entered the regional lymph nodes may also benefit from adjuvant treatment (Voll et al., 2014). To more accurately individualize prognosis and plan appropriate adjuvant therapy, additional tissue-based prognostic indicators have been sought on a molecular level. A large number of molecular, protein, and carbohydrate markers have been investigated as possible prognostic factors, but none have as yet been validated for patient care (Schimanski et al., 2005). Heat-shock protein 27 kDa (HSP27) also known as heat-shock protein β-1 (HSPB1) is a protein that in humans is encoded by the HSPB1 gene (Alekseev et al., 2009). The human HSP27 gene is located in chromosome 7q11.23, and two pseudogenes are detected in the ninth and 10th chromosomes (Ellis, 2006; Richardson et al., 2006). HSP27 is released primarily from endothelial cells and regulates angiogenesis by direct interaction with vascular endothelial growth factor (Brundel et al., 2008). It was reported that matrix metalloproteinase 9 can cleave HSP27 and release antiangiogenic fragments, which play a key role in tumor progression (Choi et al., 2014). The main function of HSP27 is to provide thermotolerance in vivo, cytoprotection, and support of cell survival under stress conditions through inhibition of apoptosis (Huang et al., 2010). Increased resistance of tumors to cancer therapy induced by HSP27 overexpression seems to depend on many factors. As already mentioned, HSP27 plays an important ‘housekeeping’ role and protects the cell against different kinds of stresses (including chemotherapy) by preventing accumulation of aggregated proteins, modulating the redox state of the cell, protecting the cytoskeleton, modulating transcription, regulating apoptosis, and probably enhancing the DNA repair in cancer cells (Ciocca and Calderwood, 2005).

It is overexpressed in a wide range of human cancers and is implicated in tumor cell proliferation, differentiation, invasion, metastasis, death, and recognition by the immune system (Ciocca et al., 2013). Its concentrations may be useful biomarkers for assessing the degree of differentiation and the aggressiveness of some cancers. HSP27 expression is associated with a poor prognosis in gastric, liver and prostate carcinoma, and osteosarcomas (Qi et al., 2014). High HSP27 expression also predicts a poor response to chemotherapy in breast cancer and leukemia, and may be related to estrogen in the breast and to estrogen and progesterone in the endometrium (Fanelli et al., 2008). It has been shown that some but not all estrogen-positive breast cancers express HSP27, and its overexpression has been associated with the degree of tumor differentiation and response to hormonal therapy such as tamoxifen (Choi et al., 2014). Liu et al. (2010) suggested that HSP27 is aberrantly expressed in CRC by carrying out a comparative proteomic study between CRC tissue and adjacent noncancerous colorectal tissue; their results reported that HSP27 might be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of CRC. Also, Bauer et al. (2012) reported that high and HSP27 expression is associated with worse clinical outcome in colon cancer.

Materials and methods

Paraffin-embedded blocks of 60 archived cases of CRC tissue were obtained from the Department of Pathology, Faculty of Medicine, Tanta University, in the period between January 2010 and December 2014. Tissue sections stained with routine hematoxylin and eosin were reviewed by two pathologists to confirm the diagnosis and accuracy of the existing data. Cancer-specific data evaluated for each patient included stage at presentation, tumor grade, specific histology, tumor location, number of positive lymph nodes, and metastases. Each tumor stage was coded as described by the AJCC, 6th ed., of CRC TNM stage organization (Fleming et al., 2012). According to Yu et al. (2010), tumor grade was categorized as low grade (well or moderately differentiated) and high grade (poorly differentiated, anaplastic, or undifferentiated). Tumor location was categorized as right (cecum, ascending colon, hepatic flexure), transverse, and left (splenic flexure, descending colon). Appendiceal, carcinoma, and carcinoma in situ were excluded. The numbers of positive lymph nodes were categorized and examined.

Immunohistochemistry

The avidin–biotin complex method was used to detect the HSP27 protein expression. Formalin-fixed and paraffin-embedded tissues were deparaffinized with xylene and rehydrated in ethanol. After quenching of endogenous peroxidase activity with 3% hydrogen peroxide for 30 min, the slides were submerged in EDTA antigenic retrieval buffer (pH 8.0) and heated in a microwave oven. Sections were blocked for 30 min at 37°C with 1% BSA and rabbit polyclonal HSP27 antibody (#IW-PA1121; IHC World), ready to use, and incubated on slides in a moist chamber overnight at 4°C. Biotinylated goat antimouse secondary antibody was applied for 15 min at 37°C, followed by incubation with a complex of avidin with horseradish peroxidase for 15 min at 37°C. Sections were developed with diaminobenzidine and counterstained with hematoxylin. Finally, the slides were sequentially dehydrated in ethanol and mounted. HSP27 immunoreactivity was present mainly in the cytoplasm of tumor cells. The positive control was human breast carcinoma. The negative control was stained by replacing the primary antibody with a nonimmune isotonic antibody. Assessment of immunostaining was performed. The level of HSP27 staining in the tumors was classified into four groups (0, absent; 1, weak; 2, intermediate; and 3, strong staining) on the basis of a scoring method described previously. Negative and weak staining was identified as showing low HSP27 expression, whereas cases with intermediate and strong staining showed high HSP27 expression (Yu et al., 2010).

Statistical analysis

All statistical analyses were carried out using the SPSS package (version 13.0 for Windows; SPSS Inc.). The correlation between HSP27 expression and the clinicopathologic characteristics was determined using the χ2-test. The Spearman rank correlation was used to assess the relationships between various variables.

Results

This work included 60 cases of previously diagnosed CRC. Most of the patients were men (66.6%), with a mean age older than 60 (53.4%) years. Twenty-five cases had colon cancer and 35 cases had rectum cancer. The grading of the cases ranged from well differentiated to poorly differentiated, and TNM stage ranged from stages 0 to IV. The clinicopathological data of the studied cases are summarized in Table 1. Then, an immunohistochemical study of HSP27 was carried out. Five (8.4%) cases were immunonegative for HSP27 (Fig. 1), whereas 55 (91.6%) cases showed detectable HSP27 immunoreactivity (weak, moderate, and strong); these results are shown in Figs 2–6. According to Yu et al. (2010), negative and weak staining was considered to show low HSP27 expression (Figs 1 and 2), whereas intermediate and strong staining was considered to show high HSP27 expression (Figs 3–6). The results suggested that it was overexpressed in CRC compared with adjacent noncancerous tissue. Twenty-four (40%) cases were identified and showed weak HSP27 expression, whereas 31 (51.6%) cases with intermediate and strong staining showed high HSP27 expression. Nontumorous adjacent mucosa showed negative immunoreactivity to HSP27. Statistical analyses were carried out to examine the correlation between immunohistochemical expression of HSP27 and the clinicopathological features of the studied CRC. These results are reported in Table 2. HSP27 expression was found to be statistically correlated with TNM staging of the cases studied (P=0.004).

T1-12
Table 1:
Clinicopathological data of the studied cases
F1-12
Fig. 1:
Nontumorous adjacent normal mucosa showing negative stain for HSP27 (Immunohistochemistry, ×400).
F2-12
Fig. 2:
Well-differentiated colorectal cancer with strong cytoplasmic positivity for HSP27 (Immunohistochemistry, ×400).
F3-12
Fig. 3:
Well-differentiated colorectal cancer with moderate cytoplasmic stain for HSP27 (Immunohistochemistry, ×200).
F4-12
Fig. 4:
Moderately differentiated colorectal cancer with weak cytoplasmic positivity for HSP27 (Immunohistochemistry, ×400).
F5-12
Fig. 5:
Moderately differentiated colorectal cancer with strong positivity for HSP27 (Immunohistochemistry, ×400).
F6-12
Fig. 6:
Poorly differentiated colorectal cancer with strong positivity for HSP27 (Immunohistochemistry, ×400).
T2-12
Table 2:
Correlation between immunohistochemical expression of HSP27 and clinicopathological data of the studied cases

Discussion

CRC is common, accounting for 15% of all newly diagnosed cancers, and tends to be a disease of the elderly, with the median age of diagnosis between 60 and 80 years of age. There is also a slight male predilection in rectal cancers, not found for tumors elsewhere in the colon (Fleming et al., 2012). Heat-shock proteins are protective proteins present in almost all species; they are used as biomarkers of various stress conditions in humans. Heat-shock protein member proteins, including HSP27, inhibit apoptosis by acting on the caspase-dependent pathway and against apoptosis-inducing agents such as tumor necrosis factor-α, staurosporine, and doxorubicin (Arrigo and Gibert, 2014). This role leads to its involvement in many pathological processes, such as oncogenesis (Parcellier et al., 2003). In particular, overexpression of HSP27 has been linked to the development of some cancers, such as hepatocellular carcinoma, gastric cancers, colonic tumors, breast cancers, and lung cancers, which led to its use as a prognostic marker for these cancers (Harm et al., 2009; Ghosh et al., 2013). This study aimed to evaluate the immunohistochemical expression of HSP27 in 60 cases of CRC and its correlation with clinical parameters, grading, and staging. It was found that it had a low expression in 48.4% of cases. Most of these cases with low expression were good and moderately differentiated carcinoma. However, 51.6% of the cases studied showed high HSP27 expression, most of which were moderately and poorly differentiated CRC. These results were in agreement with Nadin et al. (2012), who reported higher expression of HSP27 in poorly differentiated CRC. For TNM staging of the cases studied, HSP27 was overexpressed in 51.6% of the cases studied; these cases were of stages III and IV. Similar results were reported by Yu et al. (2010); they reported overexpression of HSP27 in stages III and IV CRC, and they found that cases in the low expression group of HSP27 had a better survival in comparison with the high HSP27 expression group. In the present work, a significant increase in the expression of HSP27 was observed in stage IV CRC with distant metastasis. These findings were not in agreement with those obtained by Voll et al. (2014), who reported increased HSP27 expression with increased metastasis as well as primary tumor mass. They explained that cell invasion was dependent on matrix metalloproteinase 2, whose expression was increased by HSP27. These findings indicated that HSP27 drives metastatic spread of cancer cells. According to the results of many previous studies as well as the results of this present work, overexpression of HSP27 was reported with advanced TNM stages of CRC. In other words, overexpression of HSP27 is observed in cases with poor prognostic factors such as advanced grades and stages. An increased expression of HSP27 in tumor cells of primary CRC was described as a negative bad prognostic factor for survival (Yu et al., 2010; Wang et al., 2012). The exact mechanisms remain poorly understood; however, this may be because of the physiological features of the tumor microenvironment, such as low glucose, low pH, and low oxygen (Foster et al., 2009). High expression of HSP27 in high grades and advanced stages of CRC could be explained by its capability of inhibiting apoptotic cell death by directly interacting with different apoptotic proteins at key regulatory points in the control of apoptosis (Alekseev et al., 2009). It has been shown that HSP27 inhibits apoptosis by increasing the antioxidant defense mechanism of cells (Ciocca et al., 2013). The antiapoptotic effects of HSP27 may underscore the potential role of HSP27 in cancer resistance to chemotherapy and radiotherapy (Arrigo and Gibert, 2014). Furthermore, studies have shown the effect of HSP27 in inhibiting radiation-induced apoptosis in several types of cancer, suggesting that inhibition of HSP27 expression acts as a radiation sensitizer in radioresistance cancer (Hadchity et al., 2009). Further researches showed that the HSP27 vaccine could elicit specific antitumor immunity. The high-level expression of HSP72 may not only be used as diagnostic or prognostic markers for gastrointestinal carcinomas but also as better immunotherapeutic vaccines in the cancers (Qi et al., 2014). HSP27, systemically, or confined to the tumor stroma, might possess potential prognostic and predictive value. Pulmonary metastasectomy is a widely offered treatment option in patients with CRC with pulmonary metastasis. However, the identification of patients who will benefit from surgery alone or an optional adjuvant chemotherapy is a subject of current research. Biomarkers such as HSP27 might help to identify patients with a high risk of early recurrence of disease after surgery. Adjuvant chemotherapy and a stringent follow-up could be offered to these patients (Schweiger et al., 2014).

Conclusion

HSP27 may play an important role in cancer development, progression, and response to therapy. Overexpression of HSP27 is usually associated with a poor prognosis and decreased survival rate. Oncologists have to consider the relationship between its overexpression and resistance to chemotherapy as well as radiotherapy as it supports cell survival under stress conditions.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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